Non-toxic Polymeric Dots with the Strong Protein-Driven Enhancement of One- and Two-Photon Excited Emission for Sensitive and Non-destructive Albumin Sensing

The need for efficient probing, sensing, and control of the bioactivity of biomolecules (e.g., albumins) has led to the engineering of new fluorescent albumins’ markers fulfilling very specific chemical, physical, and biological requirements. Here, we explore acetone-derived polymer dots (PDs) as promising candidates for albumin probes, with special attention paid to their cytocompatibility, two-photon absorption properties, and strong ability to non-destructively interact with serum albumins. The PDs show no cytotoxicity and exhibit high photostability. Their pronounced green fluorescence is observed upon both one-photon excitation (OPE) and two-photon excitation (TPE). Our studies show that both OPE and TPE emission responses of PDs are proteinaceous environment-sensitive. The proteins appear to constitute a matrix for the dispersion of fluorescent PDs, limiting both their aggregation and interactions with the aqueous environment. It results in a large enhancement of PD fluorescence. Meanwhile, the PDs do not interfere with the secondary protein structures of albumins, nor do they induce their aggregation, enabling the PD candidates to be good nanomarkers for non-destructive probing and sensing of albumins.


PDs' notations
Two hydrophilic fractions of acetone-derived PDs are considered in this work:  C1Na denotes PDs received from sodium hydroxide-mediated (NaOH) synthesis;  C1K corresponds to PDs fabricated using potassium hydroxide (KOH) as a catalyst.
More details regarding the synthesis and purification processes along with fundamental characterizations of their structural and linear optical properties. 1 Figure S1. Normalized absorption (black) and fluorescence (blue) spectra of native albumins.            Three excitation wavelengths are also indicated. Figure S16. Log I vs. log P for protein-nanostructure assemblies.

The fluorescence enhancement coefficients
The fluorescence enhancement coefficients were estimated from OPE and TPE emission spectra, according to the following equation: where FEC(λ exc. ) denotes the fluorescence enhancement coefficient (%), I complex and I free PDs represent the integrated fluorescence intensity for a protein-nanostructure complex and unbound PDs (a.u.), respectively.

Absolute fluorescence quantum yields
To quantify how albumins improve fluorescence properties of PDs, the absolute fluorescence quantum yields (FQYs) were estimated, as shown below: 3-4 where FQY is the calculated fluorescence quantum yield (%), S 2 denotes the integrated fluorescence intensity of sample (a.u.), S 0 and S 1 are the integrated intensity of an excitation peak in the absence and presence of a sample (a.u.), respectively, S 3 denotes the background in the emission region (a.u.);

Two-photon absorption cross-section and brightness
Figure S17. Molar-weight scaled TPA cross-section spectra of free and protein-including C1Na systems and their corresponding two-photon brightness spectra.
All fluorescence decays were fitted with commercially available analysis software (SPCImage, Becker&Hickl), using the tri-exponential equation as follows: where I(t) is the fluorescence intensity (a.u.), t represents time (ns), τ 1 , τ 2 , τ 3 denote lifetime components, and A 1 , A 2 , and A 3 are the relative decay amplitudes.  UV circular dichroism (CD) spectra of albumins and their corresponding bioconjugates at different luminophore/protein ratios were recorded, keeping constant concentration of albumins. To stimulate the inert environment, the sample chamber was deoxygenated with dry nitrogen, these conditions were held during the experiment. Spectroscopic measurements were performed, following the guidance provided by Norma J. Greenfield. 5 Additionally, pure PDs' dispersions were also measured to verify their chiroptical activity. Each CD spectrum were averaged after five accumulations. Afterward, as-obtained CD spectra were analysed with the K2D3 software to compute the secondary protein structure of native and bound albumins, as described by Caroline Louis-Jeune et al. 6 . Figure S21. Changes in CD spectra of free proteins upon the titration with PDs dispersions.
Evolution of the secondary protein structure as a function of PDs concentration.

Fourier-transform infrared spectra
The attenuated total-reflectance Fourier-transform infrared spectra (ATR-FTIR) of native proteins, pure PDs, and their conjugates were recorded in two different media, such as: Milli-Q and heavy water. All spectroscopic measurements and post-data treatment (incl. blank sample and nanostructure corrections) were performed, as reported in Huayan Yang et al. 7 Figure S22. Normalized Amide I and II bands of native proteins and conjugates. The ATR-FTIR spectra in the wavenumber region.    is the hydrodynamic diameter (nm) and SD denotes the standard deviation (nm).   Raw NMR data were interpreted based on the peaks' positions in the NMR spectroscopy handbooks, [8][9] identifying the most crucial sub-units. Aliphatic H-C Figure S31. The 13 C NMR spectrum of C1Na. The major sub-groups of PDs are indicated with their peak positions above.    Table S9. Regression curve characteristics, where a denotes the slope of the linear relation between the integrated fluorescence intensity of PDs and the concentration of albumins, σ is the standard error of the regression line. Both parameters were rounded.